Recherche Images Maps Play YouTube Actualités Gmail Drive Plus »
Connexion
Les utilisateurs de lecteurs d'écran peuvent cliquer sur ce lien pour activer le mode d'accessibilité. Celui-ci propose les mêmes fonctionnalités principales, mais il est optimisé pour votre lecteur d'écran.

Brevets

  1. Recherche avancée dans les brevets
Numéro de publicationUS5780588 A
Type de publicationOctroi
Numéro de demandeUS 08/009,296
Date de publication14 juil. 1998
Date de dépôt26 janv. 1993
Date de priorité26 janv. 1993
État de paiement des fraisCaduc
Autre référence de publicationCA2114156A1, CA2114156C, DE69407322D1, DE69407322T2, EP0612762A1, EP0612762B1
Numéro de publication009296, 08009296, US 5780588 A, US 5780588A, US-A-5780588, US5780588 A, US5780588A
InventeursGeorge R. Pettit, Jayaram K. Srirangam
Cessionnaire d'origineArizona Board Of Regents
Exporter la citationBiBTeX, EndNote, RefMan
Liens externes: USPTO, Cession USPTO, Espacenet
Elucidation and synthesis of selected pentapeptides
US 5780588 A
Résumé
The sea hare Dolabella auricularia has yielded many structurally distinct peptides which possess antineoplastic activity. Presently the compound denominated "dolastatin 10" represents the most important of such peptides because of its demonstrated potential as an anticancer drug.
The present invention relates to the systematic creation of five unique pentapeptides by selectively coupling a tripeptide-trifluoroacetate salt with a preselected dipeptide-trifluoroacetate salt which provide active molecules capable of emulating the measured therapeutic effect of dolastatin 10. The pentapeptides hereof have the structure shown below: ##STR1## wherein R is selected from the following group of substituents: ##STR2##
Images(12)
Previous page
Next page
Revendications(19)
What is claimed:
1. A composition of matter having the structural formula designated 3(a-e): ##STR5## wherein R is selected from the following group of substituents: ##STR6##
2. A composition of matter according to claim 1 in which R is the substituent designated a.
3. A composition of matter according to claim 1 in which R is the substituent designated b.
4. A composition of matter according to claim 1 in which R is the substituent designated c.
5. A composition of matter according to claim 1 in which R is the substituent designated d.
6. A composition of matter according to claim 1 in which R is the substituent designated e.
7. A method for inhibiting the growth of human cancer cells wherein said cancer is selected from the group consisting of leukemia, ovarian cancer, CNS cancer, mammary cancer, non-small cell lung cancer, renal cancer, colon cancer, and melanoma consisting of administering an active ingredient selected from the group consisting of: Dov-Val-Dil-Dap-Phe-OCH.sub.3, Dov-Val-Dil-Dap-Phe-NH.sub.2, Dov-Val-Dil-Dap-Pro-OCH.sub.3, Dov-Val-Dil-Dap-Ile-OCH.sub.3, and Dov-Val-Dil-Dap-Met-OCH.sub.3, to said cells in a quantity sufficient to inhibit the growth of said cells.
8. A method according to claim 7 wherein said active ingredient consists of Dov-Val-Dil-Dap-Phe-OCH.sub.3.
9. A method according to claim 7 wherein said active ingredient consists of Dov-Val-Dil-Dap-Phe-NH.sub.2.
10. A method according to claim 7 wherein said active ingredient consists of Dov-Val-Dil-Dap-Pro-OCH.sub.3.
11. A method according to claim 7 wherein said active ingredient consists of Dov-Val-Dil-Dap-Ile-OCH.sub.3.
12. A method according to claim 7 wherein said active ingredient consists of Dov-Val-Dil-Dap-Met-OCH.sub.3.
13. A method according to claim 7 for inhibiting the growth of human cancer cells selected from the group of cell lines consisting of P388 Lymphotic Leukemia, L1210 Lumphatic Leukemia, B16 Melanoma, M5076 Ovary Sarcoma, LOX Human Melanoma, Human Mammary MX-7, and OVCAR-3, consisting of administering an active ingredient selected from the group consisting of Dov-Val-Dil-Dap-Phe-OCH.sub.3, Dov-Val-Dil-Dap-Phe-NH.sub.2, Dov-Val-Dil-Dap-Pro-OCH.sub.3, Dov-Val-Dil-Dap-Ile-OCH.sub.3, and Dov-Val-Dil-Dap-Met-OCH.sub.3, to said cells in a quantity sufficient to inhibit the growth of said cells.
14. A method according to claim 7 wherein said cancer is selected from the group of cell lines consisting of P388, OVCAR-3, SF-295, A498, NCI-H460, KM20L2, and SK-MEL-3.
15. A method according to claim 14 wherein said active ingredient consists of Dov-Val-Dil-Dap-Phe-OCH.sub.3.
16. A method according to claim 14 wherein said active ingredient consists of Dov-Val-Dil-Dap-Phe-NH.sub.2.
17. A method according to claim 14 wherein said active ingredient consists of Dov-Val-Dil-Dap-Pro-OCH.sub.3.
18. A method according to claim 14 wherein said active ingredient consists of Dov-Val-Dil-Dap-Ile-OCH.sub.3.
19. A method according to claim 14 wherein said active ingredient consists of Dov-Val-Dil-Dap-Met-OCH.sub.3.
Description

To further aid in the understanding of the present invention, and not by way of limitation, the following examples are presented.

EXAMPLE Ia

Synthesis of Boc-Dap-Phe-OCH.sub.3 (1a)

The general procedure for the synthesis of dipeptides (1a-1e) was followed. The numerical identificate shown in Scheme 1 is followed herein. Chromatographic separation on a SILICA GEL column with 3:1 hexane-acetone as the eluent resulted in the required dipeptide as a thick oil. Crystallization from ether-hexane gave sparkling crystals of the pure compound (1a, 96%); m.p.=125 0.41, CHCl.sub.3); IR(thin film): 3314, 2974, 2934, 2878, 1748, 1692, 1663, 1537, 1456, 1400, 1366, 1173, 1101 and 700; .sup.1 H NMR (300 MHz, CDCl.sub.3): 1.163(d, J=7.0 Hz, 3H, CH.sub.3), 1.4816(s, 9H, t-Bu), 1.624-1.850(m, 4H, 2 J=13.9 and 7.8 Hz, 1H, 1/2 CH.sub.2 --Ph), 3.175(dd, J=13.8 and 5.55 Hz, 1H, 1/2 CH.sub.2 --Ph), 3.3642(s, 3H, OCH.sub.3), 3.3701(s, 3H, OCH.sub.3), 3.50-3.60(m, 1H, CH--OCH.sub.3), 3.7422(m, 2H, CH.sub.2 --N), 3.85(m, 1H, pro CH--N), 4.80(m, 1H, phe CH--N), 6.10, 6.75(m, 1H, NH) and 7.10-7.32(m, 5H, Ph); MS: m/z 416(M--MeOH), 375, 316, 264, 210, 170, 114(100%) and 70. Anal. Found: H: 8.12, N: 6.20. C.sub.24 H.sub.36 N.sub.2 O.sub.6 requires H: 8.09, N: 6.25.

EXAMPLE Ib

Synthesis of Boc-Dap-Phe-NH.sub.2 (1b)

The general procedure for the synthesis of dipeptides (1a-1e) was followed. Chromatographic purification using a SILICA GEL column with 1:1 hexane-acetone as the eluent gave the required dipeptide as a crystalline solid. Recrystallization from acetone gave sparkling crystals of the pure compound (1b, 65%); m.p.=199 α!.sub.D.sup.25 =-40 (c 0.15, CHCl.sub.3); IR(thin film): 3302, 3198, 2974, 2934, 2878, 1669, 1539, 1456, 1404, 1366, 1169, 1111 and 700; .sup.1 H NMR (300 MHz, CDCl.sub.3): 1.019(brs, 3H, CH.sub.3), 1.426(s, 9H, t-Bu), 1.55-1.90(m, 4H, 2 3.00-3.25(m, 3H, CH.sub.2 --N, CH--OCH.sub.3), 3.349(s, 3H, OCH.sub.3), 3.60-3.75(m, 1H, pro CH--N), 4.60-4.80(m, 1H, phe CH--N), 5.30(brs, 1H, NH), 6.287(d, J=7.2 Hz, 1H, NH), 6.90(brm, 1H, NH) and 7.164-7.306(m, 5H, C.sub.6 H.sub.5); MS: m/z 433(M.sup.+), 401(M--MeOH), 360, 301, 247, 232, 210, 170, 154, 138, 114 and 70(100%). Anal. Found: C: 63.75, H:8.18, N: 9.62. C.sub.23 H.sub.35 N.sub.3 O.sub.5 requires C: 63.72, H: 8.14, N: 9.69.

EXAMPLE Ic

Synthesis of Boc-Dap-Pro-OCH.sub.3 (1c)

The general procedure for the synthesis of dipeptides was followed. Chromatographic separation on a SILICA GEL column with 3:2 hexane-acetone as the eluent gave the required dipeptide as a thick oil (1c, 92%); α!.sub.D.sup.25 =-101.5 (c 0.2, CHCl.sub.3); IR(neat): 2974, 2880, 1748, 1692, 1647, 1398, 1366, 1171 and 1098; .sup.1 H NMR (300 MHz, CDCl.sub.3): 1.222(d, J=7.0 Hz, 3H, CH.sub.3), 1.440(s, 9H, t-Bu), 1.65-2.20(m, 8H, 4 1H, CH--OCH.sub.3), 3.417(s, 3H, CH.sub.3), 3.45-3.65(m, 4H, 2 and 4.447(dd, J=8.55 and 3.5 Hz, 1H, CH--COOCH.sub.3). HRFABMS: m/z 399.24880(M+H).sup.+ ;. C.sub.20 H.sub.35 N.sub.2 O.sub.6 !.sup.+ requires 399.24951.

EXAMPLE Id

Synthesis of Boc-Dap-Ile-OCH.sub.3 (1d)

The general procedure for the synthesis of dipeptides (1a-1e) was followed. Chromatographic purification on a SILICA GEL column with 3:2 hexane-ethyl acetate as the eluent yielded the required dipeptide as an oily liquid (1d, 72%); m.p.=76 =-28.2 (c 0.17, CHCl.sub.3); IR(thin film): 3325, 2971, 2936, 2878, 1746, 1694, 1667, 1530, 1478, 1398, 1254, 1175, 1105, 868 and 774; .sup.1 H NMR (300 MHz, CDCl.sub.3): 0.882(d, J=6.9 Hz, 3H, CH.sub.3 --CH), 0.9012(t, J=7.4 Hz, 3H, CH.sub.3 --CH.sub.2), 1.05-1.24(m, 5H, CH.sub.3, CH.sub.2 --CH.sub.3), 1.4526(s, 9H, t-Bu), 1.65-2.00(m, 5H, 2 CH--CH.sub.2), 2.30-2.50(m 1H, CH--CO), 3.18-3.28(m, 1H, CH--OCH.sub.3), 3.422(s, 3H, OCH.sub.3), 3.48-3.60(m, 1H, pro CH--N), 3.699(s, 3H, OCH.sub.3), 3.72-3.82(m, 1H, 1/2 CH.sub.2 --N), 3.88-3.98(m, 1H, 1/2 CH.sub.2 --N, 4.44-4.58(m, 1H, ile CH--N) and 6.15, 6.7(m, 1H, NH); MS: m/z 382(M--MeOH), 341, 282, 245, 230, 210, 170, 114, 70(100%) and 57. Anal. Found: C: 61.06, H: 9.25, N: 6.64. C.sub.21 H.sub.38 N.sub.2 O.sub.6 requires C: 60.84, H: 9.24, N: 6.76.

EXAMPLE Ie

Synthesis of Boc-Dap-Met-OCH.sub.3 (1e)

The general procedure for the synthesis of dipeptides (1a-1e) was followed. Chromatographic separation on a SILICA GEL column using 3:2 hexane-acetone as the eluent gave the required dipeptide as a solid (1e, 83%); m.p.=68 CHCl.sub.3 IR(neat): 3312, 2974, 2934, 2878, 1748, 1692, 1663, 1539, 1398, 1366, 1256, 1171, 1115, 866 and 774; .sup.1 H NMR (CDCl.sub.3): 1.223(brs, 3H, CH--CH.sub.3), 1.441(brs, 9H, t-Bu), 1.6-1.2(m, 6H, 3 2.070 (s, 3H, S--CH.sub.3), 2.3-2.55(m, 3H, CH.sub.2 --S, CH--CO), 3.15-3.35 (m, 2H, N--CH.sub.2), 3.420 (s, 3H, OCH.sub.3), 3.55(m, 1H, CH--OCH.sub.3), 3.716(brs, 3H, COOCH.sub.3), 3.85-4.0(m, 1H, pro CH--N), 4.6(brm, 1H, met CH--N), 6.3(brm, 1H, NH); MS (m/z): 432 (M.sup.+), 400, 359, 258, 210, 170, 114(100%). Anal. Found: C: 55.35, H: 8.33, N: 6.53, S: 7.23. C.sub.20 H.sub.36 N.sub.2 0.sub.6 S requires C: 55.53, H: 8.39, N: 6.48, S: 7.41.

EXAMPLE IIa

Synthesis of Dap-Phe-OCH.sub.3 Tfa (2a)

General procedure A was followed. After removing toluene under reduced pressure, the residue obtained as a thick oily mass was titrated with ether to obtain the trifluoroacetate salt (2a, quantitative) as a colorless crystalline solid: IR(thin film): 3275, 2928, 1744, 1674, 1541, 1456, 1202, 1132 and 721; .sup.1 H NMR (300 MHz, CDCl.sub.3): 1.107(brs, 3H, CH.sub.3), 1.60-2.10(m, 4H, 2 2.90-3.00(m, 2H, CH.sub.2 --Ph), 3.10-3.35(m, 3H, CH--OCH.sub.3, CH.sub.2 --N), 3.209(s, 3H, OCH.sub.3), 3.40-3.55(m, 1H, pro CH--N), 3.712(s, 3H, COOCH.sub.3), 4.75(m, 1H, phe CH--N), 7.106(m, 1H, NH), 7.124-7.324(m, 5H, Ph) and 8.7(m, 1H, NH); HRFABMS: m/z 349.21350(100%, cation); C.sub.19 H.sub.29 N.sub.2 O.sub.4 !.sup.+ requires 349.21273.

EXAMPLE IIb

Synthesis of Dap-Phe-NH.sub.2 Tfa (2b)

General procedure A was followed. Removal of toluene under reduced pressure left the trifluoroacetate salt (2b, 97%) as a colorless solid.

EXAMPLE IIc

Synthesis of Dap-Pro-OCH.sub.3 Tfa (2c)

General procedure A was followed. After removing toluene under reduced pressure, residue obtained as a thick oily mass was triturated with ether to obtain the trifluoroacetate salt (2c, 99%) as a colorless crystalline solid: IR(thin film): 2980, 2890, 1746, 1680, 1626, 1437, 1287, 1200, 1094, 799 and 721; .sup.1 H NMR (300 MHz, CDCl.sub.3): 1.307(d, J=6.9 Hz, 3H, CH.sub.3), 1.85-2.30(m, 8H, 4 3.20-3.40(m, 1H, CH--OCH.sub.3), 3.485(s, 3H, CH.sub.3), 3.35-3.75(m, 3H, CH--N, CH.sub.2 --N), 3.687(s, 3H, COOCH.sub.3), 4.165(m, 2H, CH.sub.2 --N.sup.+), 4.442(m, 1H, CH--N.sup.+) and 8.008(m, NH). HRFABMS: m/z 299.19770(100%, cation); C.sub.15 H.sub.27 N.sub.2 O.sub.4 !.sup.+ requires 299.1971.

EXAMPLE IId

Synthesis of Dap-Ile-OCH.sub.3 Tfa (2d)

General procedure A was followed. After removing toluene under reduced pressure, the residue obtained as a thick oily mass was triturated with ether to obtain the trifluoroacetate salt (2d, 97%) as a gummy mass: IR(thin film): 3289, 2969, 2884, 1744, 1674, 1541, 1458, 1383, 1202, 1136, 833, 799 and 721; .sup.1 H NMR (300 MHz, CDCl.sub.3): 0.88(brs, 3H, CH.sub.3), 1.884(t, J=6.7 Hz, 3H, CH.sub.3 --CH.sub.2), 1.209(d, J=6.8 Hz, CH.sub.3 --CH), 1.10-1.50(m, 2H, CH.sub.2), 1.80-2.20(m, 5H, 2 CH.sub.2 --N), 3.470(s, 3H, OCH.sub.3), 3.60-3.70(M, 1H, CH--OCH.sub.3), 3.85-3.90(m, 1H, pro CH--N), 3.702(s, 3H, COOCH.sub.3), 4.43(dd, J=7.5 and 5.4 Hz, 1H, ile CH--N), 6.926(d, J=7.9 Hz, 1H, NH), 8.8(m, 1H, 1/2 NH.sub.2) and 10(m, 1H, 1/2 NH.sub.2); MS: HRFAB: m/z 315.22890(100%. cation); C.sub.16 H.sub.31 N.sub.2 O.sub.4 !.sup.+ requires 315.22838.

EXAMPLE IIe

Synthesis of Dap-Met-OCH.sub.3 Tfa (2e)

General procedure A was followed. Removal of toluene under reduced pressure left the trifluoroacetate salt (2e, quantitative) as a gummy mass.

EXAMPLE IIIa

Synthesis of Dov-Val-Dil-Dap-Phe-OCH.sub.3 (3a)

Chromatographic separation on a SILICA GEL column with 3:4 hexane-acetone as the eluent gave the required pentapeptide(3a, 87%); m.p.=80 CHCl.sub.3); IR(thin film): 3298, 2963, 2934, 2876, 2830, 2787, 1748, 1622, 1532, 1454, 1379, 1269, 1200, 1099, 1038, 737 and 700; MS: m/z 759(M.sup.+), 716, 481, 449, 433, 227, 186, 154, 128, 100(100%), 85 and 70. Anal. Found: C: 64.91, H: 9.33, N: 8.97. C.sub.41 H.sub.69 N.sub.5 O.sub.8 requires C: 64.71, H: 9.15, N: 9.22.

EXAMPLE IIIb

Synthesis of Dov-Val-Dil-Dap-Phe-NH.sub.2 (3b)

General procedure B was followed. Chromatographic separation on a SILICA GEL column with 1:3 hexane-acetone as the eluent resulted in the required pentapeptide as colorless powder (3b, 99%); m.p.=111 C.; α!.sub.D.sup.25 =-42 (c 0.25, CHCl.sub.3); IR(thin film): 3304, 3138, 3054, 2965, 2934, 2876, 2830, 2787, 1622, 1541, 1499, 1423, 1371, 1306, 1252, 1202, 1171, 1098, 1038, 756, 735 and 696; MS: m/z 744(M.sup.+), 701, 669, 519, 481, 418, 227, 206, 186, 170, 154, 128 and 114.

EXAMPLE IIIc

Synthesis of Dov-Val-Dil-Dap-Pro-OCH.sub.3 (3c)

General procedure B was followed. Chromatographic purification using a SILICA GEL column with 1:3 hexane-acetone as the eluent yielded the required pentapeptide as colorless powder (3c, 69%); m.p.=75 CHCl.sub.3); IR(thin film): 3293, 2963, 2876, 2830, 2789, 1750, 1624, 1422, 1385, 1273, 1198, 1096, 1040 and 733; MS: m/z 709(M.sup.+), 666, 581, 481, 449, 412, 383, 369, 297, 255, 227(100%), 199, 186, 170 and 155. Anal. Found: C: 62.51, H: 9.61, N: 9.72. C.sub.37 H.sub.67 N.sub.5 0.sub.8 requires C: 62.59, H: 9.51, N: 9.87.

EXAMPLE IIId

Synthesis of Dov-Val-Dil-Dap-Ile-OCH.sub.3 (3d)

General procedure B was followed. Chromatographic separation on a SILICA GEL column with 1:2 hexane-acetone as the eluent gave the required pentapeptide as colorless powder (3d, 80%); m.p.=80 α!.sub.D.sup.25 =-39.3 (c 0.14, CHCl.sub.3); IR(thin film): 3300, 3050, 2965, 2878, 2830, 2787, 1746, 1622, 1530, 1454, 1383, 1267, 1120, 1099, 1038 and 735; MS: m/z 725(M.sup.+), 682, 481, 399, 227, 186, 170, 154 and 128. Anal. Found: C: 63.03, H: 10.01, N: 9.77. C.sub.38 H.sub.71 N.sub.5 O.sub.8 requires C: 62.86, H: 9.86, N: 9.65.

EXAMPLE IIIe

Synthesis of Dov-Val-Dil-Dap-Met-OCH.sub.3 (3e)

General procedure B was followed. Chromatographic separation using a SILICA GEL column with 1:2 hexane-acetone as the eluent resulted in the required pentapeptide as colorless powder (3e, 78%); m.p.=63 α!.sub.D.sup.25 =-44.1 (c, 0.44, CHCl.sub.3); IR (thin film): 3297, 2963, 2934, 2876, 2830, 2787, 1750, 1620(br), 1539, 1449, 1420, 1375, 1198 and 1098; MS (m/z): 743 (M.sup.+), 700, 611, 568, 481, 417, 311, 227 and 154. Anal. Found: C: 59.78, H: 9.14, N: 9.16, S: 4.39. C.sub.37 H.sub.69 N.sub.5 O.sub.68 S requires C: 59.73, H: 9.35, N: 9.41, S: 4.31.

From the foregoing, it is readily apparent that a useful embodiment of the present invention has been herein described and illustrated which fulfills all of the aforestated objectives in a remarkably unexpected fashion. It is of course understood that such modifications, alterations and adaptations as may readily occur to the artisan confronted with this disclosure are intended within the spirit of this disclosure which is limited only by the scope of the claims appended hereto.

__________________________________________________________________________SEQUENCE LISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 5(2) INFORMATION FOR SEQ ID NO: 1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 5 amino acid residues(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE:(A) DESCRIPTION: Linear pentapeptide(iii) HYPOTHETICAL: no(iv) ANTI-SENSE: no(vi) ORIGINAL SOURCE: synthesis(ix) FEATURE:(A) NAME/KEY:  2S- 1 1R*(R*),2S!,2R* 1S*,2S*!!!-N,N-dimethyl- L-valyl-N- 2-methoxy-4- 2- 1-methoxy-2-methyl-3-oxo- 3-  2-phenyl-1-carbomethoxy!ethyl!amino!propyl!-1-pyrrolidinyl-1-(methylpropyl)-4-oxobutyl!- N-methyl-L-valinamide(B) IDENTIFICATION METHOD: by experiment usinghigh resolution nuclear magnetic resonance and massspectral techniques(C) OTHER INFORMATION: This pentapeptide iscell growth inhibitory peptide derivative(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:XaaValXaaXaaXaa(2) INFORMATION FOR SEQ ID NO: 2:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 5 amino acid residues(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE:(A) DESCRIPTION: Linear pentapeptide(iii) HYPOTHETICAL: no(iv) ANTI-SENSE: no(vi) ORIGINAL SOURCE: synthesis(ix) FEATURE:(A) NAME/KEY:  2S- 1 1R*(R*),2S!,2R* 1S*,2S*!!!-N,N-dimethyl- L-valyl-N- 2-methoxy-4- 2- 1-methoxy-2-methyl-3-oxo- 3-  2-phenyl-1-aminocarboxy!ethyl!amino!propyl!-1-pyrrolidinyl-1-(methylpropyl)-4-oxobutyl!- N-methyl-L-valinamide(B) IDENTIFICATION METHOD: by experiment usinghigh resolution nuclear magnetic resonance and massspectral techniques(C) OTHER INFORMATION: This pentapeptide iscell growth inhibitory peptide derivative(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:XaaValXaaXaaXaa(2) INFORMATION FOR SEQ ID NO: 3:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 5 amino acid residues(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE:(A) DESCRIPTION: Linear pentapeptide(iii) HYPOTHETICAL: no(iv) ANTI-SENSE: no(vi) ORIGINAL SOURCE: synthesis(ix) FEATURE:(A) NAME/KEY:  2S- 1 1R*(R*),2S!,2R* 1S*,2S*!!!-N,N-dimethyl- L-valyl-N- 2-methoxy-4- 2- 1-methoxy-2-methyl-3-oxo- 3- O-methyl-L-prolyl!propyl!-1-pyrrolidinyl- 1-(methylpropyl)-4-oxobutyl!-N-methyl-L- valinamide(B) IDENTIFICATION METHOD: by experiment usinghigh resolution nuclear magnetic resonance and massspectral techniques(C) OTHER INFORMATION: This pentapeptide iscell growth inhibitory peptide derivative(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:XaaValXaaXaaXaa(2) INFORMATION FOR SEQ ID NO: 4:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 5 amino acid residues(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE:(A) DESCRIPTION: Linear pentapeptide(iii) HYPOTHETICAL: no(iv) ANTI-SENSE: no(vi) ORIGINAL SOURCE: synthesis(ix) FEATURE:(A) NAME/KEY:  2S- 1 1R*(R*),2S!,2R* 1S*,2S*!!!-N,N-dimethyl- L-valyl-N- 2-methoxy-4- 2- 1-methoxy-2-methyl-3-oxo- 3- O-methyl-L-isoleucyl!propyl!-1-pyrrolidinyl- 1-(methylpropyl)-4-oxobutyl!-N-methyl-L- valinamide(B) IDENTIFICATION METHOD: by experiment usinghigh resolution nuclear magnetic resonance and massspectral techniques(C) OTHER INFORMATION: This pentapeptide iscell growth inhibitory peptide derivative(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:XaaValXaaXaaXaa(2) INFORMATION FOR SEQ ID NO: 5:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 5 amino acid residues(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE:(A) DESCRIPTION: Linear pentapeptide(iii) HYPOTHETICAL: no(iv) ANTI-SENSE: no(vi) ORIGINAL SOURCE: synthesis(ix) FEATURE:(A) NAME/KEY:  2S- 1 1R*(R*),2S!,2R* 1S*,2S*!!!-N,N-dimethyl- L-valyl-N- 2-methoxy-4- 2- 1-methoxy-2-methyl-3-oxo- 3-  3-thiomethyl-1-(2-carbomethoxy)ethyl!amino!propyl!-1-pyrrolidinyl-1-(methylpropyl)-4-oxobutyl!-N-methyl-L-valinamide(B) IDENTIFICATION METHOD: by experiment usinghigh resolution nuclear magnetic resonance and massspectral techniques(C) OTHER INFORMATION: This pentapeptide iscell growth inhibitory peptide derivative(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:XaaValXaaXaaXaa__________________________________________________________________________

This invention relates generally to the field of antineoplastic compounds, and more particularly to the elucidation and synthesis of selected pentapeptides prepared by coupling dipeptide salts with the known tripeptide-trifluoroacetate salt. More particularly, the present invention relates to the synthesis of five pentapeptides by the coupling of a tripeptide-trifluoroacetate salt with the respective dipeptide-trifluoroacetate salt, which was itself prepared by the coupling of dolaproine with the respective amino acid. This coupling results in compounds which are found to exhibit effective antineoplastic activity against various human cancerous tumor cell lines.

BACKGROUND OF THE INVENTION

Ancient marine invertebrate species of the Phyla Bryozoa, Molluska, and Porifera have been well established in the oceans for over one billion years. Such organisms have undergone trillions of biosynthetic reactions in their evolutionary chemistry to reach their present level of cellular organization, regulation and defense.

For example, marine sponges have changed minimally in physical appearance for nearly 500 million years. This suggests a very effective chemical resistance to evolution in response to changing environmental conditions over that period of time. Recognition of the potential for utilizing this biologically potent marine animal for medicinal purposes was recorded in Egypt about 2,700 BC and by 200 BC sea hare extracts were being used in Greece for their curative affect. This consideration along with the observation that marine animals, e.g. invertebrates and sharks, rarely develop cancer led to the systematic investigation of marine animal and plant anticancer compounds.

By 1968 ample evidence had been obtained, based on the U.S. National Cancer Institute's (NCI) key experimental cancer study systems, that certain marine organisms could provide new and antineoplastic and/or cytotoxic agents and might also lead to compounds which would be effective in the control and/or eradication of viral diseases.

Further, these marine organisms were believed to possess potentially useful drug candidates of unprecedented structure which had eluded discovery by other methods of medicinal chemistry. Fortunately, these expectations have been realized, e.g. the discovery of the bryostatins, dolastatins and cephalostatins, many of which are now in preclinical development or human clinical studies.

Those researchers presently involved in medicinal chemistry know well the time lag between the isolation of a new compound and its introduction to the market. Often this procedure takes several years and may take decades. As a result, industry, in association with the U.S. Government, has developed a system of testing criteria which serves two purposes. One is to eliminate those substances which are shown through testing to be economically counterproductive. The second, more important purpose serves to identify those compounds which demonstrate a high likelihood of success and therefore warrant the further study and qualification, and attendant expense, necessary to meet the stringent regulatory requirements which control the ultimate market place.

The current cost to develop the necessary data approaches ten million dollars per compound. As such, economics dictate that such a huge investment will be made only when there is a reasonable opportunity for it to be recovered. Absent such opportunity, there will be no investment and the research involving the discovery of these potentially life saving compounds will cease. Only two hundred years ago many diseases ravaged mankind. Many of these now have been controlled or eradicated. During the advancement of means to treat or eliminate these diseases, work with appropriate animals was of critical importance.

Current research in the control of cancer in the United States is coordinated by the National Cancer Institute (NCI). To determine whether a substance has anti-cancer properties, the NCI has established a systematic protocol. This protocol, which involves the testing of a substance against a standard cell line panel containing 60 human tumor cell lines, has been verified and has been accepted in scientific circles. The protocol, and the established statistical means for analyzing the results obtained by the standardized testing are fully described in the literature. See: Boyd, Dr. Michael R., Principles & Practice of Oncology, PPO Updates, Volume 3, Number 10, October 1989, for an in depth description of the testing protocol; and Paull, K. D., "Display and Analysis of Patterns of Differential Activity of Drugs Against Human Tumor Cell Lines; Development of Mean Graph and COMPARE Algorithm", Journal of the National Cancer Institute Reports, Vol. 81, No. 14, Page 1088, Jul. 14, 1989 for a description of the methods of statistical analysis. Both of these references are incorporated herein by this reference thereto.

Numerous substances have been discovered which demonstrate significant antineoplastic or tumor inhibiting characteristics. As stated above, many of these compounds have been extracted, albeit with great difficulty, from marine animals such as the sponge and sea hare. Once isolation and testing of these compounds has been accomplished, a practical question remains, namely how to produce commercially significant quantities of the desired substance.

Quinine, which is available in practical quantities from the bark of the cinchona plant, differs from the compounds which are extracts of marine creatures possessing antineoplastic qualities. The collection and processing of these later compounds from their natural sources ranges from grossly impractical to the utterly impossible. Ignoring the ecological impact, the population of these creatures and the cost of collection and extraction make the process unworkable. Artificial synthesis of the active compounds is the only possible solution.

Therefore, the elucidation of the structure of these antineoplastic compounds is essential. After the structure has been determined, then a means of synthesis must be determined. This is often a long and arduous procedure due to the idiosyncratic complexity of these naturally occurring, evolutionary modified compounds. In addition, research is necessary to determine whether any portion of the naturally occurring compound is irrelevant to the desired properties, so that focus can be on the simplest structure having the perceived properties.

The Constitution of the United States (Art. 1, Sec. 8) authorized Congress to establish the United States Patent and Trademark Office (USPTO) to promote scientific progress. In order to obtain patent rights, one must show the utility of the invention. Cancer cell growth in humans often causes pain, suffering, and premature death. The impairment of human cancerous tumor growth is utilitarian in that it relieves these conditions, thereby allowing the human thus affected to have a longer, more productive life. Little could be more utilitarian than this result.

The sole right obtained from the grant of a Letters Patent is to prevent others from exploiting the subject matter of the patent. This results in the protection of the inventor for a period adequate to allow the recoupment of investment. This in turn provides incentive for further research.

The recognition of antineoplastic and tumor inhibiting activity as demonstrated by accepted NCI criteria as "utility" can promote research efforts in the United States and is unequivocally essential if those efforts are to obtain even a modest modicum of success.

BRIEF SUMMARY OF THE INVENTION

The synthesis of potentially useful peptides presents one of the most essential and promising approaches to new types of anticancer and immunosuppressant drugs. The Dolastatins, an unprecedented series of linear and cyclic antineoplastic and/or cytostatic peptides isolated from Indian Ocean sea hare Dolabella auricularia represent excellent leads for synthetic modification. The very productive sea hare Dolabella auricularia has produced a number of structurally distinct peptides with excellent antineoplastic activity. Presently Dolastatin 10, a linear pentapeptide represents the most important member and is a potentially useful antineoplastic agent. Dolastatin 10 shows one of the best antineoplastic activity profiles against various cancer screens presently known.

This research has led to an effective method for the synthesis of new and very potent anti-cancer pentapeptides related in structure to Dolastatin 10. The present invention involves the structure and synthesis of five such pentapeptides as shown below. ##STR3##

Accordingly, the primary object of the subject invention is the synthesis of five pentapeptide derivatives of dolastatin 10 which exhibit effective antineoplastic activity against various human cancerous tumor cell lines.

Another object of the subject invention is the synthesis of pentapeptide derivatives of dolastatin 10 through the coupling of respective tripeptide and dipeptide trifluoroacetate salts, wherein the dipeptide salt was prepared by the coupling of dolaproine and the respective amino acid.

These and still further objects as shall hereinafter appear are readily fulfilled by the present invention in a remarkably unexpected manner as will be readily discerned from the following detailed description of an exemplary embodiment thereof.

DESCRIPTION OF THE PREFERRED EMBODIMENT

The synthesis of potentially useful peptides presents one of the most essential and promising approaches to new types of anticancer and immunosuppressant drugs. The Dolastatins, an unprecedented series of linear and cyclic antineoplastic and/or cytostatic peptides isolated from Indian Ocean sea hare Dolabella auricularia represent excellent leads for synthetic modification. The very productive sea hare Dolabella auricularia has produced a number of structurally distinct peptides with excellent antineoplastic activity. Presently Dolastatin 10, a linear pentapeptide represents the most important member and is a potentially useful antineoplastic agent. Dolastatin 10 shows one of the best antineoplastic activity profiles against various cancer screens presently known. Recently the total synthesis and absolute configuration of this structurally unique and biologically active peptide was reported. This compound has been tested in vivo and demonstrated significant activity, as shown below.

______________________________________Experimental Anticancer Activity of Dolastatin 10 inMurine in vivo Systems, T/C (μg/kg)______________________________________P388 Lymphocytic Leukemia            LOX Human Melanoma Xenographtoxic (13.0)     to (Nude Mouse)155 and 17% cures (6.5)            toxic (52)146 and 17% cures (3.25)            301 and 67% cures (26)137 (1.63)       301 and 50% cures (13)L1210 Lymphocytic Leukemia            206 and 33% cures (6.5)152 (13)         170 and 17% cures (3.25)135 (6.5)        LOX in separate experiments139 (3.25)       340 and 50% cures (43)120 (1.63)       181 and 33% cures (26)B16 Melanoma     192 (15)238 and 40% cures (11.11)            138 and 17% cures (9.0)182 (6.67)       Human Mammary Xenograph205 (4.0)        Nude Mouse171 (3.4)        Toxic (26)142 (1.44)       137 (13)M5076 Ovary Sarcoma            178 (6.25)toxic (26)       OVCAR-3 Human Ovary Xenograph166 (13)         Nude Mouse142 (6.5)        300 (40)151 (3.25)MX-1 Human MammaryXenograft(Tumor Regression)14 (52)50 (26)61 (13)69 (6.25)______________________________________

Dolastatin 10 has also been tested against a minipanel from the NCI Primary screen. These results appear below, showing the amount of Dolastatin 10 required to attain GI.sub.50 in μg/ml, against the cell lines set forth below. ##EQU1##

From the foregoing, it can be seen that the in vitro activity of dolastatin 10 in the primary screen has been confirmed by in vivo animal tests.

For the compounds disclosed in this application, the in vitro tests disclosed above are reasonably accurate predictors of anticancer activity, and not mere indicators of the desirability for further testing.

These newly discovered pentapeptide compounds (3a-3e), related to Dolastatin 10, are formed by the coupling of the respective dipeptide-fluoroacetate salts (2a-2e) with the known tripeptide-trifluoroacetate salt (4). The dipeptides (1a-1e) were in turn prepared by coupling dolaproine (5) with the respective amino acids. All compounds were characterized (physical and spectroscopic data) and tested against the murine lymphocytic P388 leukemia cell line as well as six major human cancer cell lines. The remarkable cancer cell growth inhibitory data are shown in Table 1.

                                  TABLE 1__________________________________________________________________________Potent inhibition of Cancer cell lines by pentapeptides3a-eTEST           PENTAPEPTIDEμg/ml   CELL TYPE    CELL LINE          3a   3b   3c   3d   3e__________________________________________________________________________ED.sub.50   MOUSE P388  0.0667               0.0195                    0.0088                         0.000441                              0.000389   LEUKEMIAGI-50   Ovarian    OVCAR-3          <0.0001               0.0076                    <0.0001                         <0.0001                              <0.0001   CNS   SF-295          <0.0001               0.00085                    <0.0001                         <0.0001                              <0.0001   Renal A498  <0.0001               0.00097                    <0.0001                         <0.0001                              <0.0001   Lung-NSC    NCI-H460          <0.0001               0.000095                    <0.0001                         <0.0001                              <0.0001   Colon KM20L2          <0.0001               <0.0001                    <0.0001                         <0.0001                              <0.0001   Melanoma    SK-MEL-3          <0.0001               0.00017                    <0.0001                         <0.0001                              <0.0001TGI   Ovarian    OVCAR-3          0.0011               0.0037                    <0.0001                         <0.0001                              <0.0001   CNS   SF-295          0.00017               0.049                    0.0024                         0.17 0.056   Renal A498  0.0029               0.0062                    0.0054                         <0.0001                              >1   Lung-NSC    NCI-H460          0.011               0.011                    0.0013                         0.00088                              0.13   Colon KM20L2          0.0011               0.019                    0.0022                         <0.0001                              0.00015   Melanoma    SK-MEL-3          0.00068               0.012                    <0.0001                         <0.0001                              >1LC-   Ovarian    OVCAR-3          >1   0.066                    >1   0.043                              >150 CNS   SF-295          >1   >1   >1   >1   >1   Renal A498  >1   >1   >1   >1   >1   Lung-NSC    NCI-H460          >1   >1   >1   >1   >1   Colon KM20L2          >1   0.083                    >1   >1   >1   Melanoma    SK-MEL-3          >1   >1   >1   >1   >1__________________________________________________________________________

The human cancer cell lines results shown for pentapeptides 3a-e in Table I illustrate remarkably patent and selective activity against human ovary, CNS (brain), kidney, lung, colon and melanoma type cancers. In this respect, each compound parrots a pattern previously discovered for Dolastatin 10 and as such is reasonably expected to generate in vivo data results comparable to those reported above for Dolastatin 10.

The scheme and structures of these pentapeptides appear below: ##STR4## General Procedure for the Synthesis of Dipeptides (1a-1e)

To a solution of dolaproine tfa salt (1 mmol) and the amino acid salt (1 mmol) in dry dichloromethane (2 ml), cooled to ice-bath temperature under an argon atmosphere was added dry triethylamine (3 mmol) followed by diethylcyanophophonate (1.1 mmol). The solution was stirred at the same ice bath temperature for 1-2 hr. The salts that precipitated were collected, the solvent was evaporated (under reduced pressure) and the residue chromatographed over a SILICA GEL column with solvents noted to obtain the respective dipeptides.

i) Boc-Dap-Phe-OCH.sub.3 (1a):

Chromatographic separation on a SILICA GEL column with 3:1 hexane-acetone as the eluent resulted in the required dipeptide as a thick oil. Crystallization from ether-hexane gave sparkling crystals of the pure compound (1a, 96%); m.p=125 =-15.1.degree. (c 0.41, CHCl.sub.3); IR(thin film: 3314, 2974, 2934, 2878, 1748, 1692, 1663, 1537, 1456, 1400, 1366, 1173, 1101 and 700; .sup.1 H NMR (300 MHz,CDCl.sub.3):1.163(d, J=7.0 Hz, 3H, CH.sub.3), 1.4816(s, 9H, t-Bu), 1.624-1.850(m, 4H, 2 3.045(dd, J=13.9 and 7.8 Hz, 1H, 1/2 CH.sub.2 -Ph), 3.175(dd, J=13.8 and 5.55 Hz, 1H, 1/2 CH.sub.2 -Ph), 3.3642(s, 3H, OCH.sub.3), 3.3701(s, 3H, OCH .sub.3), 3.50-3.60(m, 1H, CH-OCH.sub.3), 3.7422(m, 2H, CH.sub.2 --N), 3.85(m, 1H, pro CH--N), 4.80(m, 1H, phe CH--N), 6.10, 6.75(m, 1H, NH) and 7.10-7.32(m, 5H, Ph); MS: m/z 416 M--CH.sub.3 OH!, 375, 316, 264, 210, 170, 114(100%) and 70. Anal. Found: H: 8.12, N: 6.20. C.sub.24 H.sub.36 N.sub.2 O.sub.6 requires H:8.09, N: 6.25.

ii) Boc-Dap-Phe-Nh.sub.2 (1b):

Chromatographic purification using a SILICA GEL column with 1:1 hexane-acetone as the eluent gave the required dipeptide as a crystalline solid. Recrystallization from acetone gave sparkling crystals of the pure compound (1b, 65%); m.p.=199 α!.sub.D.sup.25 =-40 3302, 3198, 2974, 2934, 2878, 1669, 1539, 1456, 1404, 1366, 1169, 1111 and 700; .sup.1 H NMR (300 MHz, CDCl.sub.3): 1.019(brs, 3H, CH.sub.3), 1.426(s, 9H, t-Bu), 1.55-1.90(m, 4H, 2 CH--CO), 3.00-3.25(m, 3H, CH.sub.2 --N, CH--OCH.sub.3), 3.349(s, 3H, OCH.sub.3), 3.60-3.75(m, 1H, pro CH--N), 4.60-4.80(m, 1H, phe CH--N), 5.30(brs, 1H, NH), 6.287(d, J=7.2 Hz, 1H, NH), 6.90 (brm, 1H, NH) and 7.164-7.306(m, 5H, C.sub.6 H.sub.5); MS: m/z 433(M.sup.+), 401(M--MeOH), 360, 301, 247, 232, 210, 170, 154, 138, 114 and 70(100%). Anal. Found: C: 63.75, H:8.18, N:9.62. C.sub.23 H.sub.35 N.sub.3 O.sub.5 requires C: 63.72, H: 8.14, N: 9.69.

iii) Boc-Dap-Pro-OCH.sub.3 (1c):

Chromatographic separation on a SILICA GEL column with 3:2 hexane-acetone as the eluent gave the required dipeptide as a thick oil (1c, 92%); α!.sub.D.sup.25 =-101.5.degree. (c 0.2, CHCl.sub.3); IR(neat): 2974, 2880, 1748, 1692, 1647, 1398, 1366, 1171 and 1098; .sup.1 H NMR (300 MHz, CDCl.sub.3): 1.222(d, J=7.0 Hz, 3H, CH.sub.3), 1.440(s, 9H, t-Bu), 1.65-2.20(m, 8H, 4 1H, CH--OCH.sub.3), 3.417(s, 3H, CH.sub.3), 3.45-3.65(m, 4H, 2 and 4.447(dd, J=8.55 and 3.5 Hz, 1H, CH--COOCH.sub.3). HRFABMS: m/z 399.24880 M+H!.sup.+. C.sub.20 H.sub.35 N.sub.2 O.sub.6 requires 399.24951.

iv) Boc-Dap-Ile-OCH.sub.3 (1d):

Chromatographic purification on a SILICA GEL column with 3:2 hexane-ethyl acetate as the eluent yielded the required dipeptide as an oily liquid (1d, 72%); m.p.=76 =-28.2.degree. (c 0.17, CHCl.sub.3); IR(thin film): 3325, 2971, 2936, 2878, 1746, 1694, 1667, 1530, 1478, 1398, 1254, 1175, 1105, 868 and 774; .sup.1 H NMR (300 MHz, CDCl.sub.3) 0.882(d, J=6.9 Hz, 3H, CH.sub.3 --CH), 0.9012(t, J=7.4 Hz, 3H, CH.sub.3 --CH.sub.2), 1.05-1.24(m, 5H, CH.sub.3, CH.sub.2 --CH.sub.3) 1.4526(s, 9H, t-Bu), 1.65-2.00(m, 5H, 2 1H, CH--OCH.sub.3), 3.422(s, 3H, OCH.sub.3, 3.48-3.60(m, 1H, pro CH--N), 3.699(s, 3H, OCH.sub.3), 3.72-3.82(m, 1H, 1/2 CH.sub.2 --N), 3.88-3.98(m, 1H, 1/2 CH.sub.2 --N), 4.44-4.58(m, 1H, ile CH--N) and 6.15, 6.7(m, 1H, NH); MS: m/z 382(M--MeOH), 341, 282, 245, 230, 210, 170, 114, 70(100%) and 57. Anal. Found: C: 61.06, H: 9.25, N: 6.64. C.sub.21 H.sub.38 N.sub.2 O.sub.6 requires C: 60.84, H: 9.24, N: 6.76.

v) Boc-Dap-Met-OCH.sub.3 (1e):

Chromatographic separation on a SILICA GEL column using 3:2 hexane-acetone as the eluent gave the required dipeptide as a solid (1e, 83%); m.p.=68 0.59, CHCl.sub.3); IR(neat): 3312, 2974, 2934, 2878, 1748, 1692, 1663, 1539, 1398, 1366, 1256, 1171, 1115, 866 and 774; .sup.1 H NMR (CDCl.sub.3): 1.223(brs, 3H, CH--CH.sub.3), 1.441(brs, 9H, t-Bu), 1.6-1.2(m, 6H, 3 3H, CH.sub.2 --S, CH--CO), 3.15-3.35 (m, 2H, N--CH.sub.2), 3.420 (s, 3H, OCH.sub.3), 3.55(m, 1H, CH--OCH.sub.3), 3.716(brs, 3H, COOCH.sub.3), 3.85-4.0(m, 1H, pro CH--N), 4.6(brm, 1H, met CH--N ), 6.3(brm, 1H, NH); MS (m/z): 432 (M.sup.+), 400, 359, 258, 210, 170, 114(100%). Anal. Found: C: 55.35, H: 8.33, N: 6.53, S: 7.23. C.sub.20 H.sub.36 N.sub.2 O.sub.6 S requires C: 55.53, H: 8.39, N: 6.48, S: 7.41.

Synthesis of phenylalanine amide trifluoroacetate salt

To a solution of t-boc-phenylalanine amide (3, 80 mg, 0.303 mmol) in dichloromethane (0.5 ml) was added trifluoroacetic acid (1 ml) at ice-bath temperature and the solution was stirred at the same temperature for 1.5 hr. under argon atmosphere. The solvents were removed under reduced pressure and the residue taken into toluene and toluene also removed under reduced pressure to obtain a white solid of the trifluoroacetate salt (80 mg, 95%); .sup.1 H NMR (DMSO-d.sub.6, 300 MHz): 2.95-3.10(m, 2H, C.sub.6 H.sub.5 --CH.sub.2), 3.3209(brs, 2H, NH.sub.2), 3.9408(brs, 1H, CH--N), 7.236-7.317(m, 5H, C.sub.6 H.sub.5) and 7.528, 7.862, 8.150(brs, 3H, NH.sub.3.sup.+).

DEPROTECTION OF DIPEPTIDES 1a-e WITH TRIFLUOROACETIC ACID-GENERAL PROCEDURE

To a solution of the Boc-protected dipeptide (1 mmol) in dry dichloromethane (2 ml, cooled to ice-bath temperature, under an argon atmosphere) was added trifluoroacetic acid (2 ml) and the solution was stirred at the same temperature for 1-2 hr. After removing the solvent under reduced pressure, the residue was dissolved in toluene and solvent was again removed under reduced pressure. The latter operation was repeated to remove all the trifluoroacetic acid. The residue was dried (in vacuo) to obtain the trifluoroacetate salts of the respective dipeptides. Wherever possible, the trifluoroacetate salts were characterized from spectral data and physical constants recorded.

Synthesis of Dap-Phe-OCH.sub.3 Tfa (2a)

After removing toluene under reduced pressure, the residue obtained as a thick oily mass was triturated with ether to obtain the trifluoroacetate salt (2a, quantitative) as a colorless crystalline solid: IR(thin film): 3275, 2928, 1744, 1674, 1541, 1456, 1202, 1132 and 721; .sup.1 H NMR (300 MHz, CDCl.sub.3): 1.107(brs, 3H, CH.sub.3), 1.60-2.10 (m, 4H, 2 3.10-3.35(m, 3H, CH--OCH.sub.3, CH.sub.2 --N), 3.209(s, 3H, OCH.sub.3), 3.40-3.55(m, 1H, pro CH--N), 3.712(s, 3H, COOCH.sub.3), 4.75(m, 1H, phe CH--N), 7.106(m, 1H, NH), 7.124-7.324(m, 5H, Ph) and 8.7(m, 1H, NH); HRFABMS: m/z 349.21350(100%, cation); C.sub.19 H.sub.29 N.sub.2 O.sub.4 !.sup.+ requires 349.21273.

Synthesis of Dap-Phe-NH.sub.2 Tfa (2b)

Removal of toluene under reduced pressure left the trifluoroacetate salt (2b, 97%) as a colorless solid.

Synthesis of Dap-Pro-OCH.sub.3 Tfa (2c)

After removing toluene under reduced pressure, the residue obtained as a thick oily mass was triturated with ether to obtain the trifluoroacetate salt (2c, 99%) as a colorless crystalline solid: IR(thin film): 2980, 2890, 1746, 1680, 1626, 1437, 1287, 1200, 1094, 799 and 721; .sup.1 H NMR (300 MHz, CDCl.sub.3): 1.307(d, J=6.9 Hz, 3H, CH.sub.3), 1.85-2.30(m, 8H, 4 3.485(s, 3H, CH.sub.3), 3.35-3.75(m, 3H, CH--N, CH.sub.2 --N), 3.687(s, 3H, COOOCH.sub.3), 4.165(m, 2H, CH.sub.2 --N.sup.+), 4.442(m, 1H, CH--N.sup.+) and 8.008(m, NH). HRFABMS: m/z 299.19770(100%, cation); C.sub.15 H.sub.27 N.sub.2 O.sub.4 !.sup.+ requires 299.1971.

Synthesis of Dap-Ile-OCH.sub.3 Tfa (2d)

After removing toluene under reduced pressure, the residue obtained as a thick oily mass was triturated with ether to obtain the trifluoroacetate salt (2d, 97%) as a gummy mass: IR(thin film): 3289, 2969, 2884, 1744, 1674, 1541, 1458, 1383, 1202, 1136, 833, 799 and 721; .sup.1 H NMR (300 MHz, CDCl.sub.3): 0.88(brs, 3H, CH.sub.3), 1.884(t, J=6.7 Hz, 3H, CH.sub.3 --CH.sub.2), 1.209(d, J=6.8 Hz, CH.sub.3 --CH), 1.10-1.50(m, 2H, CH.sub.2), 1.80-2.20(m, 5H, 2 CH--CO), 3.10--3.41(m, 2H, CH.sub.2 --N), 3.470(s, 3H, OCH.sub.3), 3.60-3.70(M, 1H, CH--OCH.sub.3), 3.48-3.85-3.90(m, 1H, pro CH--N), 3.702(s, 3H, COOCH.sub.3), 4.43(dd, J=7.5 and 5.4 Hz, 1H, ile CH--N), 6.926(d, J=7.9 Hz, 1H, NH), 8.8(m, 1H, 1/2 NH.sub.2) and 10(m, 1H, 1/2 NH.sub.2); MS: HRFAB: m/z 315.22890(100%. Cation); C.sub.16 H.sub.31 N.sub.2 O.sub.4 !.sup.+ requires 315.22838.

Synthesis of Dap-Met-OCH.sub.3 Tfa (2e)

Removal of toluene under reduced pressure left the trifluoroacetate salt (2e, quantitative) as a gummy mass.

SYNTHESIS OF PENTAPEPTIDES 3a-e--GENERAL PROCEDURE

To a solution of the tripeptide tfa salt (4, 1 mmol) and the dipeptide tfa salt (1 mmol) in dichloromethane (2 ml, ice-bath and under argon) was added dry triethylamine (3 mmol) followed by diethylcyanophosphonate (1.1 mmol). The solution was stirred at the same temperature for 1-2hr. After removing solvent under reduced pressure the residue was chromatographed on a SILICA GEL column using the solvent system given below as eluents to obtain the respective pentapeptides (3a-e)

Dov-Val-Dil-Dap-Phe-OCH.sub.3 (3a)

Chromatographic separation on a SILICA GEL column with 3:4 hexane-acetone as the eluent gave the required pentapeptide(3a, 87%); m.p.=80 0.34, CHCl.sub.3); IR(thin film): 3298, 2963, 2934, 2876, 2830, 2787, 1748, 1622, 1532, 1454, 1379, 1269, 1200, 1099, 1038, 737 and 700; MS: m/z 759(M.sup.+), 716, 481, 449, 433, 227, 186, 154, 128, 100(100%), 85 and 70. Anal. Found: C: 64.91, H: 9.33, N: 8.97. C.sub.41 H.sub.69 N.sub.5 O.sub.8 requires C: 64.71, H: 9.15, N: 9.22.

Dov-Val-Dil-Dap-Phe-NH.sub.2 (3b)

Chromatographic separation on a SILICA GEL column with 1:3 hexane-acetone as the eluent resulted in the required pentapeptide as colorless powder (3b, 99%); m.p.=111 =-42 2934, 2876, 2830, 2787, 1622, 1541, 1499, 1423, 1371, 1306, 1252, 1202, 1171, 1098, 1038, 756, 735 and 696; MS: m/z 744(M.sup.+), 701, 669, 519, 481, 418, 227, 206, 186, 170, 154, 128 and 114.

Dov-Val-Dil-Dap-Pro-OCH.sub.3 (3c)

Chromatographic purification using a SILICA GEL column with 1:3 hexane-acetone as the eluent yielded the required pentapeptide as colorless powder (3c, 69%); m.p.=75 α!.sub.D.sup.25 =-52.7.degree. (c 0.11, CHCl.sub.3); IR(thin film): 3293, 2963, 2876, 2830, 2789, 1750, 1624, 1422, 1385, 1273, 1198, 1096, 1040 and 733; MS: m/z 709(M.sup.+), 666, 581, 481, 449, 412, 383, 369, 297, 255, 227(100%), 199, 186, 170 and 155. Anal. Found: C: 62.51, H: 9.61, N: 9.72. C.sub.37 H.sub.67 N.sub.5 O.sub.8 requires C: 62.59, H: 9.51, N: 9.87.

Dov-Val-Dil-Dap-Ile-OCH.sub.3 (3d)

Chromatographic separation on a SILICA GEL column with 1:2 hexane-acetone as the eluent gave the required pentapeptide as colorless powder (3d, 80%); m.p.=80 (c 0.14, CHCl.sub.3); IR(thin film): 3300, 3050, 2965, 2878, 2830, 2787, 1746, 1622, 1530, 1454, 1383, 1267, 1120, 1099, 1038 and 735; MS: m/z 725(M.sup.+), 682, 481, 399, 227, 186, 170, 154 and 128. Anal. Found: C: 63.03, H: 10.01, N: 9.77. C.sub.38 H.sub.71 N.sub.5 O.sub.8 requires C: 62.86, H: 9.86, N: 9.65.

Dov-Val-Dil-Dap-Met-OCH.sub.3 (3e)

Chromatographic separation using a SILICA GEL column with 1:2 hexane-acetone as the eluent resulted in the required pentapeptide as colorless powder (3e, 78%); m.p.=63 α!.sub.D.sup.25 =-44.1.degree. (c, 0.44, CHCl.sub.3); IR(thin film): 3297, 2963, 2934, 2876, 2830, 2787, 1750, 1620(br), 1539, 1449, 1420, 1375, 1198 and 1098; MS (m/z): 743 (M.sup.+), 700, 611, 568, 481, 417, 311, 227 and 154. Anal. Found: C: 59.78, H: 9.14, N: 9.16, S: 4.39. C.sub.37 H.sub.69 N.sub.5 O.sub.68 S requires C: 59.73, H: 9.35, N: 9.41, S: 4.31.

Financial assistance for this project was provided by U.S. Government Grant Number OIG-CA44344-01A1-2; the United Statess Government may own certain rights to this invention.

Citations hors brevets
Référence
1 *Bai Biochem Pharmacol, 40 , 1859, 1990.
2Bai Biochem Pharmacol, 40, 1859, 1990.
3 *Jacobsen, J. Natl Cancer Inst 83 , 1672, 1991.
4Jacobsen, J. Natl Cancer Inst 83, 1672, 1991.
Référencé par
Brevet citant Date de dépôt Date de publication Déposant Titre
US668644523 sept. 19983 févr. 2004Arizona Board Of Regents, Acting For And On Behalf Of Arizona State UniversitySynthetic antineoplastic agents derived from dolastatin 15 and methods of making same
US68848691 nov. 200126 avr. 2005Seattle Genetics, Inc.Pentapeptide compounds and uses related thereto
US70983081 nov. 200429 août 2006Seattle Genetics, Inc.Pentapeptide compounds and uses related thereto
US725625730 avr. 200214 août 2007Seattle Genetics, Inc.Pentapeptide compounds and uses related thereto
US742311612 juin 20069 sept. 2008Seattle Genetics Inc.Pentapeptide compounds and uses related thereto
US74982985 nov. 20043 mars 2009Seattle Genetics, Inc.Monomethylvaline compounds capable of conjugation to ligands
US765924131 juil. 20039 févr. 2010Seattle Genetics, Inc.Drug conjugates and their use for treating cancer, an autoimmune disease or an infectious disease
US77453943 août 200729 juin 2010Seattle Genetics, Inc.Monomethylvaline compounds capable of conjugation to ligands
US775011620 févr. 20076 juil. 2010Seattle Genetics, Inc.Antibody drug conjugate metabolites
US782953120 mars 20099 nov. 2010Seattle Genetics Inc.Drug conjugates and their use for treating cancer, an autoimmune disease or an infectious disease
US785143718 nov. 200914 déc. 2010Seattle Genetics Inc.Drug conjugates and their use for treating cancer, an autoimmune disease or an infectious disease
US79101084 juin 200722 mars 2011Incyte CorporationSheddase inhibitors combined with CD30-binding immunotherapeutics for the treatment of CD30 positive diseases
US79645663 août 200721 juin 2011Seattle Genetics, Inc.Monomethylvaline compounds capable of conjugation to ligands
US79645673 août 200721 juin 2011Seattle Genetics, Inc.Monomethylvaline compounds capable of conjugation to ligands
US796868717 oct. 200828 juin 2011Seattle Genetics, Inc.CD19 binding agents and uses thereof
US79941353 août 20079 août 2011Seattle Genetics, Inc.Monomethylvaline compounds capable of conjugation to ligands
US824225217 mai 201114 août 2012Seattle Genetics, Inc.CD19 binding agents and uses thereof
US828835214 nov. 200516 oct. 2012Seattle Genetics, Inc.Auristatins having an aminobenzoic acid unit at the N terminus
US83439287 juil. 20061 janv. 2013Seattle Genetics, Inc.Monomethylvaline compounds having phenylalanine side-chain replacements at the C-terminus
US841025024 mars 20102 avr. 2013Genentech, Inc.Anti-FGFR3 antibodies and methods using same
US851270719 avr. 201020 août 2013Seattle Genetics, Inc.Methods of treating drug-resistant cancers
US855778029 avr. 201115 oct. 2013Seattle Genetics, Inc.Monomethylvaline compounds capable of conjugation to ligands
US860910518 mars 200917 déc. 2013Seattle Genetics, Inc.Auristatin drug linker conjugates
US863764129 juil. 201128 janv. 2014Xencor, Inc.Antibodies with modified isoelectric points
US20080249030 *30 mars 20069 oct. 2008Pierre PotierAge Inhibitors
EP2100619A120 févr. 200416 sept. 2009Seattle Genetics, Inc.Anti-CD70 antibody-drug conjugates and their use for the treatment of cancer and immune disorders
EP2260858A25 nov. 200415 déc. 2010Seattle Genetics, Inc.Monomethylvaline compounds capable of conjugation to ligands
EP2289559A120 févr. 20042 mars 2011Seattle Genetics, Inc.Anit-CD70 antibody-drug conjugates and their use for the treatment of cancer and immune disorders
EP2305716A230 nov. 20056 avr. 2011Curagen CorporationAntibodies directed to gpnmb and uses thereof
EP2325208A114 déc. 200625 mai 2011Genentech, Inc.Polyubiquitin antibodies
EP2386655A210 sept. 200716 nov. 2011Genentech, Inc.Methods and compositions for the diagnosis and treatment of lung cancer using KIT or KDG gene as genetic marker
EP2446904A229 mai 20072 mai 2012Genentech, Inc.Anti-CD22 antibodies, their immunoconjugates and uses thereof
EP2447282A229 mai 20072 mai 2012Genentech, Inc.Anti-CD22 Antibodies, their Immunoconjugates and uses thereof
EP2468772A216 mars 200727 juin 2012Genentech, Inc.Antibodies to EGFL7 and methods for their use
EP2478912A15 nov. 200425 juil. 2012Seattle Genetics, Inc.Auristatin conjugates with anti-HER2 or anti-CD22 antibodies and their use in therapy
EP2486933A15 nov. 200415 août 2012Seattle Genetics, Inc.Monomethylvaline compounds conjugated with antibodies
EP2489364A15 nov. 200422 août 2012Seattle Genetics, Inc.Monomethylvaline compounds onjugated to antibodies
EP2511299A119 avr. 200617 oct. 2012Seattle Genetics, Inc.Humanized anti-CD70 binding agents and uses thereof
WO1999015130A2 *23 sept. 19981 avr. 1999Flahive Erik JSynthetic antineoplastic agents derived from dolastatin 15 and methods of making same
WO2007127506A219 janv. 20078 nov. 2007Genentech IncAnti-ephrinb2 antibodies and methods using same
WO2008141044A27 mai 200820 nov. 2008Genentech IncCysteine engineered anti-muc16 antibodies and antibody drug conjugates
WO2008150525A13 juin 200811 déc. 2008Genentech IncAnti-notch1 nrr antibodies and methods using same
WO2009046407A26 oct. 20089 avr. 2009Zymogenetics IncB7 FAMILY MEMBER zB7H6 AND RELATED COMPOSITIONS AND METHODS
WO2010039832A130 sept. 20098 avr. 2010Genentech, Inc.Anti-notch2 antibodies and methods of use
WO2010111254A123 mars 201030 sept. 2010Genentech, Inc.Novel anti-alpha5beta1 antibodies and uses thereof
WO2010141902A24 juin 20109 déc. 2010Novartis AgMETHODS FOR IDENTIFICATION OF SITES FOR IgG CONJUGATION
WO2010148223A217 juin 201023 déc. 2010Facet Biotech CorporationAnti-vegf antibodies and their uses
WO2011057120A15 nov. 201012 mai 2011Genentech, Inc.Methods and composition for secretion of heterologous polypeptides
WO2011059762A128 oct. 201019 mai 2011Abbott Biotherapeutics Corp.Anti-egfr antibodies and their uses
WO2011070443A18 déc. 201016 juin 2011Institut National De La Sante Et De La Recherche MedicaleMonoclonal antibodies that bind b7h6 and uses thereof
WO2011084496A115 déc. 201014 juil. 2011Abbott Biotherapeutics Corp.Anti-her2 antibodies and their uses
WO2011107553A13 mars 20119 sept. 2011F. Hoffmann-La Roche AgAntibodies against human csf-1r and uses thereof
WO2011113019A211 mars 201115 sept. 2011Abbott Biotherapeutics Corp.Ctla4 proteins and their uses
WO2011119661A123 mars 201129 sept. 2011Genentech, Inc.Anti-lrp6 antibodies
WO2011131407A13 mars 201127 oct. 2011F. Hoffmann-La Roche AgAntibodies against human csf-1r and uses thereof
WO2011133886A222 avr. 201127 oct. 2011Genentech, Inc.Production of heteromultimeric proteins
WO2011154453A18 juin 201115 déc. 2011Genmab A/SAntibodies against human cd38
WO2011157741A215 juin 201122 déc. 2011Genmab A/SHuman antibody drug conjugates against tissue factor
WO2011161119A121 juin 201129 déc. 2011F. Hoffmann-La Roche AgAntibodies against insulin-like growth factor i receptor and uses thereof
WO2011161189A122 juin 201129 déc. 2011F. Hoffmann-La Roche AgAnti-hepsin antibodies and methods of use
WO2012016227A229 juil. 20112 févr. 2012Xencor, Inc.Antibodies with modified isoelectric points
WO2012018771A12 août 20119 févr. 2012F. Hoffmann-La Roche AgChronic lymphocytic leukemia (cll) biomarkers
WO2012020006A29 août 201116 févr. 2012Roche Glycart AgAnti-fap antibodies and methods of use
WO2012020038A110 août 201116 févr. 2012Roche Glycart AgAnti-tenascin-c a2 antibodies and methods of use
WO2012021773A112 août 201116 févr. 2012F. Hoffmann-La Roche AgAntibodies to il-1beta and il-18, for treatment of disease
WO2012045085A13 oct. 20115 avr. 2012Oxford Biotherapeutics Ltd.Anti-rori antibodies
WO2012064836A19 nov. 201118 mai 2012F. Hoffmann-La Roche AgMethods and compositions for neural disease immunotherapy
WO2012087962A219 déc. 201128 juin 2012F. Hoffmann-La Roche AgAnti-mesothelin antibodies and immunoconjugates
WO2012088313A121 déc. 201128 juin 2012F. Hoffmann-La Roche AgAnti-pcsk9 antibodies and methods of use
WO2012092616A130 déc. 20115 juil. 2012Takeda Pharmaceutical Company LimitedConjugated anti-cd38 antibodies
WO2012106587A13 févr. 20129 août 2012Genentech, Inc.Fc VARIANTS AND METHODS FOR THEIR PRODUCTION
WO2012109624A210 févr. 201216 août 2012Zyngenia, Inc.Monovalent and multivalent multispecific complexes and uses thereof
WO2012130831A127 mars 20124 oct. 2012Roche Glycart AgAntibody fc variants
WO2012138975A16 avr. 201211 oct. 2012Genentech, Inc.Anti-fgfr4 antibodies and methods of use
WO2012143379A118 avr. 201226 oct. 2012Roche Glycart AgMethod and constructs for the ph dependent passage of the blood-brain-barrier
WO2012143523A120 avr. 201226 oct. 2012Genmab A/SBispecifc antibodies against her2
WO2012156532A121 mai 201222 nov. 2012Centre Val D'aurelle - Paul LamarqueAnti-human-her3 antibodies and uses thereof
WO2012156918A116 mai 201222 nov. 2012Koninklijke Philips Electronics N.V.Bio-orthogonal drug activation
WO2012156919A116 mai 201222 nov. 2012Koninklijke Philips Electronics N.V.Bio-orthogonal drug activation
WO2012156920A116 mai 201222 nov. 2012Koninklijke Philips Electronics N.V.Bio-orthogonal drug activation
WO2012158704A115 mai 201222 nov. 2012F. Hoffmann-La Roche AgFgfr1 agonists and methods of use
WO2012171020A111 juin 201213 déc. 2012Mersana Therapeutics, Inc.Protein-polymer-drug conjugates
WO2012175691A122 juin 201227 déc. 2012INSERM (Institut National de la Santé et de la Recherche Médicale)Anti-axl antibodies and uses thereof
WO2012175692A122 juin 201227 déc. 2012INSERM (Institut National de la Santé et de la Recherche Médicale)Anti-axl antibodies and uses thereof
WO2013004842A26 juil. 201210 janv. 2013Genmab A/SAntibody variants and uses thereof
WO2013022855A16 août 201214 févr. 2013Xencor, Inc.Antibodies with modified isoelectric points and immunofiltering
WO2013026835A121 août 201228 févr. 2013Roche Glycart AgFc-free antibodies comprising two fab fragments and methods of use
WO2013026839A121 août 201228 févr. 2013Roche Glycart AgBispecific antibodies specific for t-cell activating antigens and a tumor antigen and methods of use
WO2013041462A114 sept. 201228 mars 2013Roche Glycart AgBispecific anti-egfr/anti igf-1r antibodies
WO2013043715A119 sept. 201228 mars 2013Genentech, Inc.Combination treatments comprising c-met antagonists and b-raf antagonists
WO2013052155A16 avr. 201211 avr. 2013Genentech, Inc.Methods of treating liver conditions using notch2 antagonists
WO2013055809A110 oct. 201218 avr. 2013Xencor, Inc.A method for purifying antibodies
WO2013056054A212 oct. 201218 avr. 2013Genentech, IncPeptide inhibitors of bace1
WO2013109819A118 janv. 201325 juil. 2013Genentech, Inc.Anti-lrp5 antibodies and methods of use
WO2013109856A218 janv. 201325 juil. 2013Genentech, Inc.Methods of using fgf19 modulators
WO2013109994A118 janv. 201325 juil. 2013Sea Lane Biotechnologies, LlcSurrobody cojugates
WO2013120056A111 févr. 201315 août 2013Genentech, Inc.R-spondin translocations and methods using the same
WO2013120929A114 févr. 201322 août 2013F. Hoffmann-La Roche AgFc-receptor based affinity chromatography
WO2013170191A110 mai 201314 nov. 2013Genentech, Inc.Methods of using antagonists of nad biosynthesis from nicotinamide
WO2013174783A121 mai 201328 nov. 2013Pieris AgLipocalin muteins with binding-affinity for glypican-3 (gpc-3) and use of lipocalin muteins for target-specific delivery to cells expressing gpc-3
WO2013185115A17 juin 201312 déc. 2013Sutro Biopharma, Inc.Antibodies comprising site-specific non-natural amino acid residues, methods of their preparation and methods of their use
WO2013192360A119 juin 201327 déc. 2013Ambrx, Inc.Anti-cd70 antibody drug conjugates
WO2014001326A125 juin 20133 janv. 2014F. Hoffmann-La Roche AgMethod for the selection and production of tailor-made, selective and multi-specific therapeutic molecules comprising at least two different targeting entities and uses thereof
WO2014006118A14 juil. 20139 janv. 2014F. Hoffmann-La Roche AgAnti-theophylline antibodies and methods of use
WO2014006123A14 juil. 20139 janv. 2014F. Hoffmann-La Roche AgAnti-biotin antibodies and methods of use
WO2014006217A15 juil. 20139 janv. 2014Genmab B.V.Dimeric protein with triple mutations
WO2014036492A130 août 20136 mars 2014Sutro Biopharma, Inc.Modified amino acids comprising an azido group
Classifications
Classification aux États-Unis530/330
Classification internationaleC07K7/06, C07K1/16, C07K1/02, C07K5/02, A61P35/00, A61K38/00
Classification coopérativeC07K5/0205, A61K38/00
Classification européenneC07K5/02B
Événements juridiques
DateCodeÉvénementDescription
31 août 2010FPExpired due to failure to pay maintenance fee
Effective date: 20100714
14 juil. 2010LAPSLapse for failure to pay maintenance fees
15 févr. 2010REMIMaintenance fee reminder mailed
23 oct. 2008ASAssignment
Owner name: NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF
Free format text: CONFIRMATORY LICENSE;ASSIGNOR:ARIZONA STATE UNIVERSITY;REEL/FRAME:021726/0227
Effective date: 19930423
6 mars 2006SULPSurcharge for late payment
Year of fee payment: 7
6 mars 2006FPAYFee payment
Year of fee payment: 8
1 févr. 2006REMIMaintenance fee reminder mailed
20 déc. 2001FPAYFee payment
Year of fee payment: 4
18 janv. 1994ASAssignment
Owner name: ARIZONA BOARD OF REGENTS, A BODY CORPORATE OF THE
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:PETTIT, GEORGE R.;SRIRANGAM, JAYARAM K.;REEL/FRAME:006826/0247
Effective date: 19930125